Several of the muscular dystrophies, including the Duchenne's Becker's and Myotonic Dystrophies are characterized by abnormalities in membrane function. In Duchenne's Muscular Dystrophy, for example, intracellular calcium levels rise, resulting in a disruption of excitation-contraction (EC) coupling, the development of calcium overload and activation of Ca- dependent proteases which produce muscle necrosis. Thus, a complete understanding of muscle dysfunction necessitates an understanding of how skeletal muscle controls calcium. The ability (or inability) of the muscle to produce force depends in large part upon the release of calcium ions from the sarcoplasmic reticulum (SR) membranes of the muscle cell. During EC-coupling, calcium is released from SR through a calcium release channel called the ryanodine receptor. Our laboratory has been investigating the control of muscle contraction by endogenous sphingolipids. The major goal of the proposed research is to elucidate the mechanism skeletal muscle EC- coupling, and in particular, the role of the naturally occurring second messenger, sphingosine (SPH), and other sphingolipids in modulating calcium flux through SR membrane calcium release channels. Based on preliminary work, we hypothesize that endogenously produced SPH blocks calcium release via a direct effect on the SR calcium release channel, the ryanodine receptor, and that SPH's action may be a physiologically relevant mechanism for modulating release. We wish to test this hypothesis by examining the effects of SPH on the receptor. We have shown that SPH can substantially alter the contractile behavior of skeletal muscle cells and isolated cardiac myocyte. We have interpreted the effect of SPH as an action on intracellular calcium release channels; consequently, we intend to measure the kinetics of calcium release from isolated SR terminal cisternae (TC) vesicles in response to SPH and other sphingoid lipids. We intend to correlate SPH action on the rapid kinetics of calcium release with its effects on [3H]-ryanodine binding to isolate skeletal TC vesicles, since ryanodine binding is directly related to the level of calcium channel opening by the receptor. We will employ HPLC methods recently developed in our laboratory to assess sphingolipid levels in the muscle cytosol and various other muscle subfractions to determine if SPH and other second messengers are in the range where we observe their physiological actions on calcium release. Because our preliminary work indicates that the T-tubules of the muscle possesses the enzymatic machinery for SPH production, we plan experiments designed to measure in various muscle subfractions the presence of key enzymes involved in sphingolipid metabolism. These data will be correlated with the in situ immunocytochemical localization of SMase. Our planned experiments will determine if SPH and other sphingoid lipids are endogenous second messengers with physiological relevance in modulating contractility in striated muscle. MBRS students will be involved in all aspects of the research.
